Table_5_Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.docx

In Japan, major mumps outbreaks still occur every 4–5 years because of low mumps vaccine coverage (30–40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan’s regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

在日本，由于实施自愿性免疫规划，腮腺炎疫苗接种覆盖率仅为30%~40%，导致每4~5年仍会暴发大规模腮腺炎疫情。为做好常规免疫规划的准备工作，本研究旨在阐明日本腮腺炎病毒（mumps virus, MuV）的全国性长期分子流行病学趋势。此外，本研究采用下一代测序技术开展全基因组测序（whole-genome sequencing, WGS），以评估基于腮腺炎病毒小疏水蛋白（small-hydrophobic, SH）基因序列的常规基因分型结果的可靠性。本研究分析了1986年至2017年间，从日本25个都道府县采集的1064份腮腺炎临床样本及腮腺炎病毒分离株的SH基因序列。结果显示，共鉴定出6种基因型，分别为B型（110株）、F型（1株）、G型（900株）、H型（3株）、J型（41株）及L型（9株）；自20世纪80年代以来，日本的优势基因型每十年就会发生一次更迭。自21世纪初以来，G型始终为日本唯一流行的腮腺炎病毒基因型。基于SH基因序列分型，G型可分为7个进化分支。为验证上述结果，本研究采用下一代测序（next-generation sequencing, NGS）技术对77株G型代表性分离株进行全基因组测序，并开展系统发育分析。系统发育分析共鉴定出5个具有高自展支持值的进化分支，并将其命名为日本分支（Japanese clade, JPC）-1、-2、-3、-4及-5。JPC-1和JPC-3合计占G型分离株总数的80%以上，分别为68.3%和13.8%。其中，JPC-2和JPC-5为本研究在日本境内新鉴定的进化分支。本研究是首个针对日本腮腺炎病毒开展全国性长期分子流行病学调查的报道。本研究结果可为日本本土腮腺炎病毒基因型的相关研究提供数据支持，这对于评估日本将腮腺炎疫苗纳入常规免疫规划后，该国腮腺炎消除工作的进展具有重要意义。此外，本研究证实，采用下一代测序技术开展的全基因组测序分析，相较于传统的基于SH基因序列的分型方法，结果更为精准，可作为开展精准分子流行病学研究的有力工具。